Date of Award

1-1-2006

Degree Type

Restricted Access Thesis

Degree Name

Master of Science

Department

Graduate School of Biomedical Sciences

First Advisor

Tina K. Machu

Second Advisor

Glenn Dillon

Third Advisor

Meharavan Singh

Abstract

The 5-Hydroxytryptamine3 (the 5-HT3) receptor is composed of homomers of A subunits or heteromers of A and B subunits. The discovery of the crystal structure of the acetylcholine binding protein (Brejc et al., 2001) has helped us identify the generalized structure of the N-terminal domains of this superfamily, but the precise details regarding the amino acid residues involved in the process of ligand binding in the 5-Ht3 receptor are unknown. Mouse and human 5-HT3A receptors are 84% identical at the amino acid level, yet they have differential sensitivities to numerous drugs that bind to the ligand recognition site, for example, agonists such as 2-Methyl serotonin and m-Chlorophenyl Biguanide (m-CPBG) and competitive antagonists such as d-Tubocurarine (curare). The distal 1/3 part of the N-terminal domain, which contains both Loop C and Loop F is responsible for determining the potency of curare of the 5-HT3A receptor, these loops consist of thirteen non-conserved amino acid residues between mouse and human the 5-HT3A receptors. As a result of these differences, curare is 135-fold more potent at the mouse wild-type receptor than human wild-type receptor. In this project we utilized the differential curare sensitivity of mouse and human 5-HT3A receptors to obtain information regarding the amino acid residues in Loop C, involved in ligand binding process. Chimeric and point mutant receptors were constructed on the human receptor background with substitutions of corresponding mouse orthologs and expressed in Xenopus oocytes. Curare potency was assessed with two-electrode voltage clamp electrophysiological recordings. Our results suggests that a minimum of four mouse orthologs are required to produce the curare IC50 of 41 nM obtained in the chimeric receptor, which contains seven mouse orthologs (Hch Loop C receptor).

Comments

W 4.5 L882R 2006

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